The interplay between oligodendrocyte (OL) and extracellular matrix (ECM) is critical to the proper maturation of this unique cell type. Recent work has established the β1 integrin-signaling pathway, a mediator for ECM/OL interactions, as an essential component of myelin sheath formation in the central nervous system (CNS). A major downstream effector of β1 integrin is integrin-linked kinase (ILK), an adaptor and structural platform protein. Herein, we (1) generated a model system to study ILK in vivo and (2) employed the model to elucidate ILK’s role in regulating OL biology. To assess the importance of ILK in OL-mediated myelination, we ablated ILK in primary OLs. ILK loss delayed morphological maturation and led to filamentous actin accumulation in the processes and cell body. Further, we noted an upregulation in RhoA activity, with pathway inhibition rescuing an OL subset. We next moved our studies in vivo. First, we assessed the proteolipid protein promoter’s utility as OL-specific Cre driver. Protocols established, we generated an ILK conditional knockout line (Ilk cKO). Ultrastructural analysis of Ilk cKO optic nerves revealed increased number of amyelinated nerve fibers at P14 with subsequent recovery by P28. The observed transient defects were due neither to a loss nor a gain in total number of mature or progenitor OLs. To rationalize recovery, we grew ILK-depleted OLs on an “inert” substrate. Here, while morphology improved, ILK-depleted OLs were characterized by enlarged and sluggish growth cones as well as microtubule disorganization. Taken together, our data suggests a role for ILK in regulating the morphological maturation of OLs both in vitro and in vivo, the loss of which results in defective OL branching and membrane formation with phenotype and subsequent recovery dependent upon niche complexity.

A fundamental characteristic of neural stem cells (NSCs) is their ability to divide symmetrically, producing daughter cells of different fates. This requires the regulation of proliferation verses differentiation, which could occur in two different ways; at a cellular or environmental level. Although there is some evidence for each model, little is known about how this regulation occurs. One candidate is the integrin family. Integrins are known to regulate key aspects of stem cell behaviour. The integrin subunits α6 and β1, which heterodimerise into a laminin receptor, are highly expressed within the NSCs of the embryonic ventricular zone and loss of integrin β1 (itgβ1) function within the ventricular zone of the embryonic mouse results in NSC detachment and apoptosis. The aim of this thesis is to investigate the role of itgβ1 in the regulation of proliferation verses differentiation in the NSC of the chick embryonic neuroepithelium. To test the function of itgβ1 within this system, we are using transfection of a constitutively active (CA*), wild type (WT) and extracellular portion only (EC only) integrin-β1, via electroporation of the chick embryonic CNS, resulting in a patchwork of expression within the NSCs of the midbrain neuroepithelium. This system allows both expressing cells and their non-expressing neighbours to be studied within the same environment. We predicted that if integrins are acting to regulate NSC behaviour via an intrinsic mechanism, only the cells expressing the CA* integrin will alter their behaviour. If the second model is correct and integrins act via an extrinsic mechanism, we predicted that the neighbouring non-expressing cells will also alter their behaviour. We observed a significant increase in the number of neurons generated upon expression of CA*β1. This increase in neurons was a non-cell autonomous effect; the neurons were GFP and human itgβ1 negative, supporting the second model of extrinsic signals and cell-cell interactions in the regulation of proliferation and differentiation. The increase in neurogenesis was only observed in the midbrain upon CA*β1 expression for 48hrs. A significant increase in mitotic cells was observed 12hrs after electroporation, the earliest time point and by E4 (48hrs) a significant proportion of mitotic cells were abnormally located by 48hrs, resulting in basal mitoses. Investigation of signalling between cells was carried out using microarray analysis of the two populations of cells, CA*β1 positive and negative. One candidate from the microarray results was the bHLH transcription factor Tal2. Tal2 has previously been shown to be specifically expressed within the midbrain neuroepithelium at the time of electroporation and to play a role in the regulation of neurogenesis. In summary, this thesis has showed an important role of itgβ1 in the regulation of proliferation and differentiation of NSCs within the chick embryonic neuroepithelium in a non-cell autonomous manner.

No description available.

Integrins are heterodimeric transmembrane proteins that provide a bi-directional link between the cell’s internal biological mechanisms and the extracellular environment. During inside-out signalling, intracellular messages converge on the integrin cytoplasmic domain to induce a conformational change. This is transmitted to the extracellular domain where it results in an alteration in affinity for integrin ligands such as fibronectin and laminin. In this way the cell has developed the ability to modulate the critical functions of adhesion and cell movement. In outside-in signalling, the integrin performs a more complex function than simple adhesion; upon binding to ligand, the integrin extracellular domain undergoes a conformational change which is transmitted to the cytoplasmic domain. This alters the integrin’s cytoplasmic domain affinity for intracellular signalling proteins and results in the activation of intracellular second messenger pathways. In this way, the extracellular milieu is able to influence intracellular signalling including those involved in apoptosis. This thesis demonstrates data which provide original insights into bi-directional integrin signalling: Inside-out signalling: Constitutively active Notch1 increases β3-integrin affinity and abrogates Hras-mediated integrin suppression without increasing expression of β3- integrin. Dominant-Negative Rras blocks Notch-mediated integrin activation and Notch1-mediated reversal of Hras and Raf-mediated integrin suppression and this is independent of erk phosphorylation. Notch1 induces Rras activation. Functional adhesion assays confirm that Notch1IC increases K562 adhesion in a β1-integrin dependent manner and this is abrogated by Dominant-Negative Rras. This data supports a mechanism in which Notch1 increases integrin affinity via activation of Rras. Outside-in signalling: Evidence is presented demonstrating that extracellular matrix proteins, laminin and fibronectin, activate β1-integrins to protect SCLC cells against the apoptotic effects of etoposide and ionizing radiation via PI3Kinase activation. This occurs in two ways: 1) PI3Kinase-dependent β1-integrin signalling resulting in phosphorylation of Bad and reduced caspase-9 cleavage and 2) a β1-integrinmediated over-riding of etoposide and radiotherapy-induced cell cycle S phase delay and G2/M arrest. β1-integrin-mediated outside-in survival signalling was investigated further in the in vivo setting; MatrigelTM, a basement membrane product rich in extracellular matrix proteins, promoted SCLC xenograft survival and growth in a β1-integrin and tyrosine kinase-dependent manner. This data provides novel insights into the critical functions that integrins play in adhesion and survival signalling.

Integrins are a large family of transmembrane cell adhesion receptors that are found on the surface of eukaryotic cells. Integrins act predominantly as cell surface receptors for extracellular matrix (ECM) proteins, but also have bidirectional signaling properties that allow them to play fundamental roles in development and cancer metastasis. It has become clear that during gastrulation – a period during which cells participate in morphogenetic movements that lead to the generation of a tripoblastic embryo – the integrin repertoire of each cell is in constant flux. This change in cell surface receptors is mediated through intracellular pathways, which in turn, are regulated by associations with cytoplasmic proteins. One such molecule, GIPC (GAIP-interacting protein, C-terminus), is thought to have a role in regulating α5β1 integrin surface expression, as well as integrin-mediated inside-out and outside-in signaling pathways by mediating the integrin’s ability to interact with the ECM protein, fibronectin (FN). I use Xenopus laevis as my experimental model system to study GIPC-regulated integrin function. Xenopus provides a useful model system for regulation of integrin function as α5β1-FN interactions are spatially and temporally regulated. Additionally Xenopus embryos are amenable to molecular manipulations in vivo, and the tissue can be excised from embryos and cultured in vitro. I have investigated the function of GIPC using site-directed mutagenesis to alter the PDZ domain site in Xenopus GIPC (XGIPC). Expression of dominant negative XGIPC results in the interruption of gastrulation movements in the early embryo. Yeast two-hybrid and co-immunoprecipitation assays demonstrate that XGIPC physically interacts with the cytoplasmic domain of the α5 and α6 integrin subunit. Furthermore, I have determined that the interaction of XGIPC with α5β1 is required for assembly of a FN matrix. Cell migration and convergent extension assays demonstrate that XGIPC likely plays other undefined roles in modulating α5β1 function. XGIPC was found to be required for efficient trafficking of α5β1, as determined by α5β1 internalization assays in A6 cells. Together, my data indicate a critical role for XGIPC in modulating α5β1 integrin function during early embryonic morphogenesis.

Integrins are a large family of transmembrane cell adhesion receptors that are found on the surface of eukaryotic cells. Integrins act predominantly as cell surface receptors for extracellular matrix (ECM) proteins, but also have bidirectional signaling properties that allow them to play fundamental roles in development and cancer metastasis. It has become clear that during gastrulation – a period during which cells participate in morphogenetic movements that lead to the generation of a tripoblastic embryo – the integrin repertoire of each cell is in constant flux. This change in cell surface receptors is mediated through intracellular pathways, which in turn, are regulated by associations with cytoplasmic proteins. One such molecule, GIPC (GAIP-interacting protein, C-terminus), is thought to have a role in regulating α5β1 integrin surface expression, as well as integrin-mediated inside-out and outside-in signaling pathways by mediating the integrin’s ability to interact with the ECM protein, fibronectin (FN). I use Xenopus laevis as my experimental model system to study GIPC-regulated integrin function. Xenopus provides a useful model system for regulation of integrin function as α5β1-FN interactions are spatially and temporally regulated. Additionally Xenopus embryos are amenable to molecular manipulations in vivo, and the tissue can be excised from embryos and cultured in vitro. I have investigated the function of GIPC using site-directed mutagenesis to alter the PDZ domain site in Xenopus GIPC (XGIPC). Expression of dominant negative XGIPC results in the interruption of gastrulation movements in the early embryo. Yeast two-hybrid and co-immunoprecipitation assays demonstrate that XGIPC physically interacts with the cytoplasmic domain of the α5 and α6 integrin subunit. Furthermore, I have determined that the interaction of XGIPC with α5β1 is required for assembly of a FN matrix. Cell migration and convergent extension assays demonstrate that XGIPC likely plays other undefined roles in modulating α5β1 function. XGIPC was found to be required for efficient trafficking of α5β1, as determined by α5β1 internalization assays in A6 cells. Together, my data indicate a critical role for XGIPC in modulating α5β1 integrin function during early embryonic morphogenesis.

Stroke is the leading cause of long term disability and the third leading cause of death in the United States. Perlecan plays a significant role in brain development by sequestering and delivering growth factors to developing neuronal precursor cells in a neurovascular niche. Previous results demonstrated that perlecan proteolysis results in the cleavage of perlecan’s most C-terminal domain five (DV) in the post-ischemic brain. As post-stroke angiogenesis is an important step in post-stroke brain repair, I focused on the mechanism of DV’s role in brain angiogenesis in vitro. I first demonstrated that DV significantly increased brain endothelial (BE) cell migration, proliferation and tube-like formation suggesting DV is a pro-angiogenic factor for BE cells. I next investigated VEGF secretion from BE cells in the presence of DV. DV significantly increased VEGF secretion into the cell media, which was both dose and time dependent. Using quantitative real-time PCR, DV induced a maximal nine-fold increase in VEGF expression, compared to control, indicating DV is an upstream regulator of VEGF transcription. DV treated cells show an increase in phosphorylation of ERK-(1/2) that could be blocked by the pharmacological inhibitor U0126. This inhibitor could also block DV’s effect on VEGF mRNA expression and secretion indicating ERK is involved with DV’s effect on VEGF regulation. Optical sensor binding assays confirmed that DV binds to the α5β1 integrin with a Kd of 160nM, and cells treated with DV showed a visual representation of integrin α5β1-DV colocalization. Furthermore, shRNA-mediated knockdown of integrin α5 blocked DV’s effect on VEGF mRNA expression, indicating integrin α5 is involved with DV’s regulation of VEGF expression. In conclusion, these results demonstrate that DV has an unexpected proangiogenic effect in brain angiogenesis. This occurs via a previously unreported interaction between DV and the α5β1 integrin, resulting in the activation of the ERK, eIF4A and HIF1α signaling pathway and an ultimate increase in VEGF mRNA expression and VEGF secretion. As DV is generated post-stroke, these results suggest a novel mechanism by which brain tissue recovery following ischemia is influenced by processed fragments from the extracellular matrix.

Fibronectin (Fn) is an adhesive extracellular matrix protein assembled by fibroblasts into fibrils within ECMs of developing and remodeling tissues. Fn is sensitive to mechanical forces exerted by contractile cells, and can alter its structural conformations in response to mechanical strain within Fn fibrils. We developed probes (both peptide and antibody) to Fn to detect mechano-sensitive perturbations of Fn conformation. Probes were characterized for their binding characteristics (affinity, epitope, mechano-sensitivity) and validated on multiple in vitro and in vivo ECM models. Furthermore, we showed that the mechano-sensitive H5 antibody that we have developed have utility in detection of early molecular signatures of fibrosis in vivo in a mouse model of pulmonary fibrosis. Using the H5 antibody, we also report detection of a conformational switch within the integrin binding FnIII9-10 region. Modulation of Fn’s integrin switching behavior may help in the development of controllable “smart” biomaterials, as well as to the development of conformation-specific imaging probes to detect early molecular signatures of tumor and fibrotic ECMs.

Leukocyte exit from peripheral tissues is fundamental to host defense, yet little is known about the role of adhesive molecules in this process. In my thesis I ask the question “can an integrin regulate leukocyte exit from inflamed peripheral tissues” and specifically investigate the leukocyte integrin α1β1. This is an important question because leukocyte exit, or persistence, at an inflammatory lesion can have a profound effect on the immune response. In addition, I present special in situ staining techniques which had to be developed in order to assay endogenous leukocyte migration in a murine model. The introductory sections review functional differences between myeloid and lymphoid leukocyte subsets, the leukocyte adhesion cascade, integrins, chemokine receptors and the essential concepts of signaling and the relationship between chemokines and integrin activation. I also discuss the pro-migratory paradigm of leukocyte integrins, in other words that integrin adhesion is equated with leukocyte migration. The current literature regarding what is known about integrin function in peripheral tissues and leukocyte migration is also discussed. Chapter 2 characterizes my inflammatory model and implicates α1β1integrin and macrophages as important molecular and cellular entities respectively, involved in sustaining the inflammatory response. Chapter 3 develops endogenous in-situ labeling in the blood compartment, establishing the fundamentals of my in-situ approach. Chapter 4 extends this and establishes in-situ pulse labeling (ISPL) to label endogenous leukocytes in peripheral tissues. Chapter 4 then goes on to combine the technological advances and conceptual framework established in the previous chapters to elucidate a role for α1β1integrin in the exit of macrophages from inflamed peripheral tissues. Finally, in Chapter 5 I discuss the implications of my results in the context of the host defense, how it might impact the immune response and future directions for this research.

Leukocyte exit from peripheral tissues is fundamental to host defense, yet little is known about the role of adhesive molecules in this process. In my thesis I ask the question “can an integrin regulate leukocyte exit from inflamed peripheral tissues” and specifically investigate the leukocyte integrin α1β1. This is an important question because leukocyte exit, or persistence, at an inflammatory lesion can have a profound effect on the immune response. In addition, I present special in situ staining techniques which had to be developed in order to assay endogenous leukocyte migration in a murine model. The introductory sections review functional differences between myeloid and lymphoid leukocyte subsets, the leukocyte adhesion cascade, integrins, chemokine receptors and the essential concepts of signaling and the relationship between chemokines and integrin activation. I also discuss the pro-migratory paradigm of leukocyte integrins, in other words that integrin adhesion is equated with leukocyte migration. The current literature regarding what is known about integrin function in peripheral tissues and leukocyte migration is also discussed. Chapter 2 characterizes my inflammatory model and implicates α1β1integrin and macrophages as important molecular and cellular entities respectively, involved in sustaining the inflammatory response. Chapter 3 develops endogenous in-situ labeling in the blood compartment, establishing the fundamentals of my in-situ approach. Chapter 4 extends this and establishes in-situ pulse labeling (ISPL) to label endogenous leukocytes in peripheral tissues. Chapter 4 then goes on to combine the technological advances and conceptual framework established in the previous chapters to elucidate a role for α1β1integrin in the exit of macrophages from inflamed peripheral tissues. Finally, in Chapter 5 I discuss the implications of my results in the context of the host defense, how it might impact the immune response and future directions for this research.